Development of surface modified biodegradable polymeric nanoparticles to deliver GSE24.2 peptide to cells: a promising approach for the treatment of defective telomerase disorders.

The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.

Reactivation of plasma butyrylcholinesterase by pralidoxime chloride in patients poisoned by WHO class II toxicity organophosphorus insecticides.

Some clinicians assess the efficacy of pralidoxime in organophosphorus (OP) poisoned patients by measuring reactivation of butyrylcholinesterase (BuChE). However, the degree of BuChE inhibition varies by OP insecticide, and it is unclear how well oximes reactivate BuChE in vivo. We aimed to assess the usefulness of BuChE activity to monitor pralidoxime treatment by studying its reactivation after pralidoxime administration to patients with laboratory-proven World Health Organization (WHO) class II OP insecticide poisoning. Patient data were derived from 2 studies, a cohort study (using a bolus treatment of 1g pralidoxime chloride) and a randomized controlled trial (RCT) (comparing 2g pralidoxime over 20 min, followed by an infusion of 0.5 g/h, with placebo). Two grams of pralidoxime variably reactivated BuChE in patients poisoned by 2 diethyl OP insecticides, chlorpyrifos and quinalphos; however, unlike acetylcholinesterase reactivation, this reactivation was not sustained. It did not reactivate BuChE inhibited by the dimethyl OPs dimethoate or fenthion. The 1-g dose produced no reactivation. Pralidoxime produced variable reactivation of BuChE in WHO class II OP-poisoned patients according to the pralidoxime dose administered, OP ingested, and individual patient. The use of BuChE assays for monitoring the effect of pralidoxime treatment is unlikely to be clinically useful.

Dose-ranging study with the glucokinase activator AZD1656 in patients with type 2 diabetes mellitus on metformin.

AIM: To investigate the effect of glucokinase activator AZD1656 on glycated haemoglobin (HbA1c) as an add-on to metformin in patients with type 2 diabetes. METHODS: This randomized, double-blind, placebo-controlled study (NCT01020123) was conducted over 4 months with an optional 2-month extension. Patients (n = 458) with HbA1c 7.5-10% were randomized to AZD1656 20 mg (n = 40) or 40 mg (n = 52) fixed doses or 10-140 mg (n = 91) or 20-200 mg (n = 93) titrated doses, placebo (n = 88) or glipizide 5-20 mg titrated (n = 94). Patients (n = 72) with HbA1c >10 and ≤12% received open-label AZD1656 (20-200 mg titrated). Primary outcome was placebo-corrected change in HbA1c from baseline to 4 months of treatment. RESULTS: Significant reductions in HbA1c from baseline to 4 months were observed with blinded AZD1656 10-140 and 20-200 mg versus placebo [mean (95% CI) changes: -0.80 (-1.14; -0.46) and -0.81 (-1.14; -0.47) %, respectively), with similar reductions observed with glipizide. A higher percentage of patients on AZD1656 than on placebo achieved HbA1c ≤7.0 or ≤6.5 % after 4 months. Mean (s.d.) change in HbA1c for open-label AZD1656 (20-200 mg) was -2.8 (1.19) % after 4 months. AZD1656 was well tolerated, with less hypoglycaemia than glipizide. In the extension population, HbA1c was still reduced with AZD1656 versus placebo after 6 months, but the effect of AZD1656 on glucose control was not sustained over time. CONCLUSION: Addition of AZD1656 (individually titrated) to metformin gave significant improvements in glycaemic control up to 4 months, although efficacy diminished over time.

Reactivating PP2A by FTY720 as a novel therapy for AML with C-KIT tyrosine kinase domain mutation.

The tyrosine kinase domain (TKD) mutations of receptor tyrosine kinase C-KIT are associated with a poor prognosis in acute myeloid leukemia (AML). However, the underlying mechanisms are not fully understood. We found the activity of protein phosphatase 2A (PP2A), a human tumor suppressor whose dysfunction contributes to malignant cell behavior, was significantly decreased in AML subgroups harboring C-KIT/D816V and AML cell line Kasumi-1 bearing C-KIT/N822K mutation. Primary AML cells and various AML cell lines were treated with PP2A activator FTY720. FTY720 showed a toxic effect in all leukemic cells, especially for cells harboring C-KIT/TKD mutation. Furthermore, FTY720-induced toxicity in AML leukemic cells was mediated by restoration of PP2A activity, via down-regulation of PP2A inhibitor SET, dephosporylation of PP2A-C(TYR307), and up-regulation of relevant PP2A subunit A and B55α. Our research indicates that the decreased PP2A activity in AML harboring C-KIT/TKD mutation may make the restoration of PP2A activity a novel therapy for AML patients with C-KIT/TKD mutation.

New bispyridinium oximes: in vitro and in vivo evaluation of their biological efficiency in soman and tabun poisoning.

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still a challenge for the scientific community. This study investigated the interactions of four bispyridinium oximes with human erythrocyte acetylcholinesterase (AChE) and their effects on soman- and tabun-poisoned mice. Oximes HI-6 and TMB-4 were used for comparison. These oximes inhibited AchE with inhibitory potency (IC(50)) ranging from 0.02 to 1.0 mM. The best reactivating potency (%R) was obtained with K074, when AChE was inhibited by tabun. The protective potency (P(50)) of all oximes in human erythrocyte AChE inhibited by soman and tabun could not be determined. In tabun-poisoned mice very good antidotal efficacy was obtained with K027, K048, and K074, which makes them interesting for future investigation. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.

Oximes: Reactivators of phosphorylated acetylcholinesterase and antidotes in therapy against tabun poisoning.

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime, found as the most effective reactivators, have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE. Three of our tested pyridinium oximes K027, K048, K074, along with TMB-4, were the most promising for AChE reactivation. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Herein, we showed that a promising treatment in tabun poisoning by selected oximes and atropine could be improved if oximes are also used in pretreatment. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.

Síndrome de Hurler-Scheie: seguimiento de la terapia de recambio enzimático durante dos años / No disponible

No disponible

A Rho-kinase inhibitor, soluble guanylate cyclase activator and nitric oxide-releasing PDE5 inhibitor: novel approaches to erectile dysfunction.

Approximately 50% of men aged over 40 suffer from male erectile dysfunction. Treatment options have widened since the launch of the phosphodiesterase type 5 (PDE5) inhibitor, sildenafil citrate (Viagra trade mark ). However, a certain portion of the patient population, such as diabetics, do not gain significant benefit from PDE5 inhibitors, possibly due to a lack of endogenous nitric oxide. Therefore, new treatment modalities based on the absence of endogenous nitric oxide have been developed. Among them are Rho-kinase inhibitors, soluble guanylate cyclase activators and nitric oxide-releasing PDE5 inhibitors. The available data concerning these compounds will be summarised and their therapeutic potential for male erectile dysfunction will be discussed.

In vivo reactivation of DNases in implanted human prostate tumors after administration of a vitamin C/K(3) combination.

Human prostate cancer cells (DU145) implanted into nude mice are deficient in DNase activity. After administration of a vitamin C/vitamin K(3) combination, both alkaline DNase (DNase I) and acid DNase (DNase II) activities were detected in cryosections with a histochemical lead nitrate technique. Alkaline DNase activity appeared 1 hr after vitamin administration, decreased slightly until 2 hr, and disappeared by 8 hr after treatment. Acid DNase activity appeared 2 hr after vitamin administration, reached its highest levels between 4 and 8 hr, and maintained its activity 24 hr after treatment. Methyl green staining indicated that DNase expression was accompanied by a decrease in DNA content of the tumor cells. Microscopic examination of 1-microm sections of the tumors indicated that DNase reactivation and the subsequent degradation of DNA induced multiple forms of tumor cell death, including apoptosis and necrosis. The primary form of vitamin-induced tumor cell death was autoschizis, which is characterized by membrane damage and the progressive loss of cytoplasm through a series of self-excisions. These self-excisions typically continue until the perikaryon consists of an apparently intact nucleus surrounded by a thin rim of cytoplasm that contains damaged organelles.

A comparison of the efficacy of acetylcholinesterase reactivators against cyclohexyl methylphosphonofluoridate (GF agent) by in vitro and in vivo methods.

The purpose of this study was to compare the therapeutic efficacy of a new acetylcholinesterase reactivator, designated BI-6 (1-(2-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium )-2-butene dibromide), with presently used oximes (pralidoxime, obidoxime, methoxime) and H-oximes (HI-6, HLö-7) by in vitro and in vivo methods. In vitro, methoxime seems to be the most efficacious reactivator of GF agent-inhibited acetylcholinesterase because the phosphorylation of acetylcholinesterase by GF agent markedly increases its affinity for the enzyme. The oxime BI-6 is more efficacious than other presently used oximes (pralidoxime, obidoxime) but its reactivating efficacy does not reach the efficacy of H-oximes tested. On the other hand, obidoxime and pralidoxime appear to be very poor reactivators of GF agent-inhibited acetylcholinesterase because the phosphonylation of acetylcholinesterase by GF agent markedly decreases their affinity to the enzyme. In vivo, H oximes (HI-6, HLö-7) are the most efficacious antidotes for the treatment of acute poisoning with GF agent in rats while the presently used oximes such as pralidoxime and obidoxime are practically ineffective. BI-6 and methoxime are more efficacious than pralidoxime and obidoxime, nevertheless their therapeutic efficacy does not reach the efficacy of H oximes. Our results show that the ability of oximes to reactivate GF agent-inhibited acetylcholinesterase in vitro usually corresponds to their therapeutic effects against GF agent in vivo.

[Methods of treating experimental Neguvon poisoning and a comparison of their effects]. / Zpusoby lécení experimentální otravy Neguvonem a srovnání jejich úcinku.

The effect of conventional antidotal therapy of oral intoxication due to organophosphorus insecticide Neguvon in male mice is compared to the effect of peritoneal dialysis. As a dialysate, the combination of ACHE reactivator with atropine, distilled water and ACHE preparation has been used. In order to evaluate treatment results, the clinical course of intoxication and the increase of blood ACHE activity inhibited with perorally Neguvon were investigated. The highest increase in activity of inhibited blood ACHE has been stated by the peritoneal dialysis with ACHE preparation. Furthermore, the effects of two dialysates were compared. The first one consisted in ACHE reactivator with atropine, and the second one in ACHE preparation, respectively. The ACHE containing dialysate showed better therapeutical results. The mentioned acetylcholinesterase is stated to be inhibited through the treatment with 50% of Neguvon dosage applied to the experimental animals. Thus the actual dose of Neguvon inducing proper intoxication was reduced to its half amount.

[Structure-activity relationship in reactivators of acetylcholinesterase inhibited by phosphoric esters. XII. Hydroxyiminomethylalkylpyridine iodomethylate]. / Relazione struttura-attività nei riattivatori dell'acetilcolinesterasi inibita da esteri fosforici. XII. Idrossiminometilalchilpiridine iodometilate.

2-Hydroxyiminomethyl-4-alkylpyridine methiodides were prepared and tested for their in vitro reactivating potency on phosphorylated electric eel cholinesterase. They were also tested as in vivo protecting agents: only one of the compounds, 4-methyl-2-hydroxyiminopyridine, offered a greater protection from DFP than 2-PAM, the others had little to no effect.